Role of Organelle-localized Lys63-Linked Ubiquitination in Plant Immunity

全世界每年因植物病害造成的作物产量损失高达数十亿美元，这对养活全球迅速增长的人口的粮食安全构成了令人震惊的威胁。因此，了解并最终利用植物抵御各种病原体的关键分子机制来改良作物，具有重要的农业、经济和社会意义。宿主防御的关键机制之一是泛素化，这是一种主要的翻译后蛋白修饰过程，发生在植物、动物和人类细胞中。通过泛素（赖氨酸-63连接泛素化）对蛋白质进行的一种特殊类型的非常规修饰，在调节人类和动物的先天和适应性免疫中起着至关重要的作用。然而，这种类型的泛素化在植物免疫和其他植物生理过程中的作用尚未得到充分研究，尽管这种泛素化是植物中第二丰富的泛素化类型。本项目研究了这种非常规泛素化调节植物先天免疫的机制基础，包括对高中科学教师和少数民族本科生的教育发展，以及对内布拉斯加州农民和利益相关者的宣传。本项目初步鉴定并初步暗示了两个番茄环型泛素连接酶Fti1和Fti1B及其在拟南芥中最接近的同源物AtFti1和AtFti1B分别参与fls2介导的植物模式触发免疫（PTI）。研究发现，Fti1和Fti1B及其拟南芥同源物与UBC13型泛素结合酶相互作用，催化K63-linked泛素化，Fti1B和AtFti1B在fls2介导的PTI中功能保守。此外，在细胞中flg22诱导PTI后，发现AtFti1B的一个异构体将亚细胞定位改变为一个尚未确定的细胞器。在这个项目中，研究人员将阐明通过AtFti1B/ fti1b介导的K63-linked泛素化调控植物PTI的分子机制，目的是：(1)确定AtFti1B亚型在PTI中的作用以及AtFti1B- bak1 /FLS2相互作用的分子结果；(2)阐明AtFti1B- bak1 /FLS2相互作用、AtFti1B的E3连接酶活性以及AtFti1B在植物免疫中的亚细胞定位的生物学意义；(3)鉴定atfti1b介导的Lys63泛素化调控植物免疫的新组分。该项目的完成将有助于解决植物免疫中两个重要但难以捉摸的问题：lys63连接的泛素化和细胞器在植物免疫信号传导中的作用和机制。考虑到目前对Lys63-linked ubiquitination在植物生物学中的认知有限，本项目的研究结果也将有助于我们了解Lys63-linked ubiquitination在其他植物生理过程中的作用。该项目将包括广泛的教育和推广活动，通过高中和本科生科学联系促进对研究的浓厚兴趣（HUSKER）计划，培训在科学领域代表性不足的高中科学教师和少数民族本科生，并教育内布拉斯加州农民和利益相关者关于作物改良基础研究的重要性。

Crop yield losses caused by plant diseases are in the value of billions of dollars worldwide each year, which has made it an alarming threat to the food security in feeding a burgeoning global population. Understanding and eventually manipulating for crop improvement the key molecular mechanisms which plants use to defend against various pathogens is thus of considerable agricultural, economic, and social importance. Among the mechanisms pivotal for host defense is ubiquitination, a major post-translational protein modification process that occurs in plant, animal, and human cells. A particular type of unconventional modification of proteins by ubiquitin (lysine-63-linked ubiquitination, is well documented to be crucial in modulating human and animal innate and adaptive immunity. However, the roles of this type of ubiquitination in plant immunity and other plant physiological processes have been significantly understudied, even though this type of ubiquitnation is the second most abundant type of ubiquitination in plants. This project investigates the mechanistic basis of regulating plant innate immunity by this type of unconventional ubiquitination and includes education development for high school science teachers and minority undergraduate students and outreach to the Nebraska farmers and stakeholders.Initial work of this project has identified and preliminarily implicated two tomato RING type ubiquitin ligases, Fti1 and Fti1B and their closest homolog in Arabidopsis, AtFti1 and AtFti1B, respectively in FLS2-mediated plant pattern-triggered immunity (PTI). Fti1 and Fti1B and their Arabidopsis closest homolog were found to interact and work with UBC13 type ubiquitin-conjugating enzyme to catalyze K63-linked ubiquitination and Fti1B and AtFti1B were shown to be functionally conserved in FLS2-mediated PTI. Additionally, an isoform of AtFti1B was found to change subcellular localization to a yet undetermined organelle after PTI was induced by flg22 in the cell. In this project the investigators will elucidate the molecular mechanism underlying the regulation of plant PTI by AtFti1B/Fti1B-mediated K63-linked ubiquitination, with aims to (1) determine the role of AtFti1B isoforms in PTI and the molecular outcome of AtFti1B-BAK1/FLS2 interaction; (2) elucidate the biological significance of AtFti1B-BAK1/FLS2 interaction, the E3 ligase activity of AtFti1B, and the AtFti1B subcellular localization in plant immunity; and (3) identify novel components that are involved in the regulation of plant immunity by AtFti1B-mediated Lys63 ubiquitination. Fulfillment of this project will help integrally address two important, yet elusive questions in plant immunity: what are the roles and mechanisms of Lys63-linked ubiquitination and cell organelles in plant immune signaling. Considering the limited knowledge of Lys63-linked ubiquitination in plant biology in general, findings from this project will also assist our efforts to understand Lys63-linked ubiquitination in other plant physiological processes. Integral to this project will be extensive education and outreach activities to train high school science teachers and minority undergraduate students that are underrepresented in science through a High School and Undergraduate Science Connect Promoting Keen Interests in Research (HUSKER) program and to educate Nebraska farmers and stakeholders about the importance of basic research in crop improvement.